International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220

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International Journal for Parasitology: Parasites and Wildlife

journal homepage: www.elsevier.com/locate/ijppaw

Large (Nematoda: ) recovered from the European bison may represent a new subspecies

Anna M. Pyziel a,*, Zdzisław Laskowski b, Izabella Dolka c, Marta Kołodziej-Sobocinska´ d, Julita Nowakowska e, Daniel Klich f, Wojciech Bielecki g, Marta Zygowska˙ a, Madeleine Moazzami h, Krzysztof Anusz a, Johan Hoglund¨ i a Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS–SGGW), Department of Food Hygiene and Public Health Protection, Nowoursynowska 159, 02-776, Warsaw, Poland b Polish Academy of Sciences, W. Stefanski´ Institute of Parasitology, Twarda 51/55, 00-818, Warsaw, Poland c Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS–SGGW), Department of Pathology and Veterinary Diagnostics, Division of Pathology, Nowoursynowska 159c, 02-776, Warsaw, Poland d Mammal Research Institute, Polish Academy of Sciences, Stoczek 1, 17-230, Białowieza,˙ Poland e Institute of Biology, University of Warsaw, Laboratory of Electron & Confocal Microscopy, Miecznikowa 1, 20-096, Warsaw, Poland f Institute of Animal Sciences, Warsaw University of Life Sciences (WULS-SGGW), Department of Animal Genetics and Conservation, Ciszewskiego 8, 02-787, Warsaw, Poland g Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Department of Pathology and Veterinary Diagnostics, Division of Avian Diseases, Exotic and Fish, Ciszewskiego 8, 02-786, Warsaw, Poland h Swedish University of Agricultural Sciences (SLU), Department of Biomedical Sciences and Veterinary Public Health (BVF), Division of Bacteriology and Food Safety, Box 7035, 75007, Uppsala, Sweden i Swedish University of Agricultural Sciences (SLU), Department of Biomedical Sciences and Veterinary Public Health (BVF), Division of Parasitology, Box 7035, 75007, Uppsala, Sweden

ARTICLE INFO ABSTRACT

Keywords: Although the , the agent of dictyocaulosis, is one of parasitological threats to European Dictyocaulus viviparus bison, its systematic position remains unclear. The aim of the present study was to evaluate the morphological Bison bonasus features of the lungworm and the pathological lesions it induces, and to analyse mitochondrial (mt) genetic Morphological description markers for systematic and molecular epidemiological studies. The morphological findings indicate that Dic­ Verminous pneumonia tyocaulus lungworms of European bison can be distinguished from those of on the basis of differences in Mitochondrial genetic markers buccal capsule wall length, total body length, and spicules length in males, all of which were significantlylonger in those of European bison. Nucleotide diversity calculated from pairwise sequence alignments of partial cyto­ chrome c oxidase subunit 1 (cox1), cytochrome B (cytB) and NADH dehydrogenase subunit 5 (nad5) of specimens from cattle and European bison varied from 1.7% for nad5, 2.1% for cytB, to 3.7% for cox1 gene. Thus, among the lungworms of European bison and cattle, nad5 and cytB were the most conserved proteins, whereas cox1 was the most diverse. The mt cytB marker gene may be a suitable candidate for distinguishing between the two geno­ types, as nad5 demonstrated the greatest within- sequence variation. The lung tissue of infected European bison manifests signs of verminous pneumonia characterized by interstitial pneumonia, bronchitis and bron­ chiolitis. Therefore, it appears that European bison and cattle are infected with slightly diverged, morphologically-different, genotypes of D. viviparus, indicating they belong to two separate worm populations. We propose, therefore, that the lungworm of European bison should be classified as D. viviparus subsp. bisontis.

1. Introduction inhabiting Poland is the European bison, Bison bonasus: the largest terrestrial mammal in Europe; however, it is considered vulnerable to Free-roaming populations of wild enrich the biodiversity extinction by the International Union for Conservation of Nature - Red of heavily-urbanized landscapes of European countries. A unique bovid List of Threatened (IUCN, 2020). The European bison also

* Corresponding author. E-mail address: [email protected] (A.M. Pyziel). https://doi.org/10.1016/j.ijppaw.2020.10.002 Received 1 September 2020; Received in revised form 7 October 2020; Accepted 7 October 2020 Available online 24 October 2020 2213-2244/© 2020 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A.M. Pyziel et al. International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220 suffers from a loss of genetic variation, and is hence more vulnerable to stored in acetone (Eisenback, 1985). The dehydrated specimens were pathogens due to inbreeding depression and the depletion of variation in then subjected to critical-point drying with liquid CO2, their proximal the genes responsible for immunity against parasites (Keller and Waller, endings were cut and mounted on a SEM mounting stub with 2002; Radwan et al., 2010). Dictyocaulosis is a condition affecting double-coated tape, sputter-coated with gold, and examined with the various ungulate hosts, characterized by symptoms of bronchitis, with use of a LEO 1430VP scanning electron microscope. infection leading fatal outcomes in heavily-infected individuals (Demi­ aszkiewicz et al., 2009; Pyziel et al., 2018a). In order to understand the 2.3. DNA extraction, amplification and sequencing risk for cross-transmission between free-roaming European bison and cattle, and to the control the spread of dictyocaulosis, it is essential to Depending on individual intensity of infection, 1–5 male worms from have the potential to correctly identify lungworm species. However, each examined European bison were used for molecular analyses. although some initial studies on European bison-derived lungworms Genomic DNA was extracted individually from 48 adult male lung­ have been published, their systematic position remains unclear (Pyziel, worms (preserved in 70% ethanol) using a Nucleospin tissue DNA 2014, 2018; Pyziel et al., 2017). extraction kit (Macherey-Nagel, Düren, Germany) according to the The small subunit (SSU) rDNA sequence of D. viviparus from Euro­ manufacturer’s protocol. Partial regions of mitochondrial (mt) cox1, pean bison has been found to be generally identical to those from cattle; NADH dehydrogenase subunit 5 (nad5), and cytochrome B (cytB) of the however, the two groups were found to diverge at the internal tran­ worms were amplified by PCR using the following sets of primers: ′ ′ scribed spacer (ITS2) region of the ribosomal RNA-array (Pyziel, 2014). LCO1490 (5 -GGT CAA CAA ATC ATA AAG ATA TTG G-3 ) + HC02198 ′ ′ Additionally, the mt cytochrome c oxidase subunit 1 (cox1) nucleotide (5 -TAA ACT ATC GGG TGA CCA AAA AAT CA-3 ) for cox1 (Folmer ′ ′ sequence demonstrates high intraspecific diversity between the two et al., 1994); OP424 (5 -ATG ACT AAT CGT ATT GGG GA-3 ) + OP425 ′ ′ strains of D. viviparus (Pyziel et al., 2017). However, the mt cytochrome (5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 ) for nad5 (Hoglund¨ ′ ′ c oxidase subunit 3 (cox3) gene was found as an inefficient mt marker et al., 2006); cytB_F (5 -TGA AAA RGT TAA GAT RRT TGG GAC-3 ) + ′ ′ for systematic study on Dictyocaulus spp. (Pyziel et al., 2018b). cytB_R (5 -TTA GGA ATA GCA CGC AAA ATA GC-3 ) for cytB (present The present study examines the morphological features of European study). The primers were designed with the use of FastPCR software, bison lungworm, evaluates its impact on the lung tissue of the host, and version 5.4 (Primer Digital, Helsinki, Finland). identifies the most efficient mt genetic markers for systematic and mo­ PCR was performed in a 2720 thermal cycler (Applied Biosystems, lecular epidemiological studies. Foster City, California) in a volume of 50 μl. Each 50 μl PCR reaction contained 20 μl of Molecular Biology Reagent Water (Sigma-Aldrich, 2. Materials and methods USA), 25 μl Quant-Bio’s AccuStart™ II PCR ToughMix® ( × 2 concen­ tration) (Quantabio, Beverly, USA), 1 μl GelTrack Loading Dye ( × 50 2.1. Specimen collection concentration) (Quantabio, Beverly, USA), 1 μl forward primer (20 mM), 1 μl reverse primer (20 mM), and 2 μl of template DNA. The conditions ◦ A total of 30 male and 35 female adult Dictyocaulus lungworms were for PCR were as follows: 94 C for 2 min to denature the DNA, with 35 ◦ ◦ isolated from the respiratory tract of 15 free-roaming European bison. cycles at 94 C for 40 s (cox1 and nad5) or for 45 s (cytB), 57 C for 45 s ◦ ◦ All European bison had been euthanized for various health reasons in (cox1 and nad5) or 56 C for 60 s (cytB), and 72 C for 30 s (cox1, nad5) Białowieza˙ Primeval Forest (n = 8) and Borecka Forest (n = 7) (north- or for 45 s (cytB); with a finalextension of 5 min (cox1, nad5) or 10 min ◦ east Poland) during the years 2017–2019. Lethal control is an element of (cytB) at 72 C to ensure complete amplifications.The PCR product was health monitoring conducted annually. Individuals of weak condition purified with the use of the Nucleospin Gel and PCR Clean-up Kit with general or specific signs of disease (e.g. necrotic inflammation of (Macherey-Nagel, Germany), eluted with 30 μl of Molecular Biology the prepuce) were selected. The formal aspects of conducted annual Reagent Water (Sigma-Aldrich, USA) and sequenced in both directions lethal control was described in Klich et al. (2020). The respiratory tracts by Macrogen Europe (Amsterdam, the Netherlands) with the use of were taken exclusively from animals infected by Dictyocaulus sp. The primers used for amplification (5 mM). The sequences were then trachea, bronchi, and bronchioles were cut open and immerses into assembled into contigs using CodonCode Aligner version 8.0 (Codon­ beakers filled with tap water, the sediment was investigated under a Code Corporation, Massachusetts, USA). The generated sequences were stereomicroscope and worms were recovered with the use of dissecting then compared with those from the same regions in D. viviparus of cattle, needles (Pyziel et al., 2017). Lungworms from each animal were stored and those downloaded from GeneBank (see below). The accession separately, preserved in 70% ethanol and then transported to the numbers are listed in Table 1. Swedish University of Agricultural Sciences for morphological and molecular investigations.

2.2. Morphological study Table 1 Specimens were cleared in lactophenol and mounted in glycerin jelly List of taxa included in the molecular analysis using combined sequence data of + + on slides following Pyziel et al. (2017). Selected morphological features mitochondrial cox1 cytB nad5. were measured: body length, buccal capsule (BC), buccal capsule wall Species Host cox1 cytB nad5

(BCW), head, cephalic vesicle, esophagus of males and females; copu­ GenBank GenBank GenBank latory bursa, gubernaculum, spicules of male reproductive system; Dictocaulus Bos taurus JX519460.1 JX519460.1 JX519460.1 vestibules, anterior sphincter and infundibulum, posterior sphincter and viviparus infundibulum, mature and immature eggs in the uterus, tail and phas­ Dictocaulus Bos taurus AP017683.1 AP017683.1 AP017683.1 mids of the female reproductive system. Additionally, location of nerve viviparus ring and excretory pore from anterior extremity were evaluated, as well Dictocaulus Bison KM359417.1 KM503300.1 KM359431.1 as location of vulva opening from tail-tip and body width at vulva viviparus bonasus Dictocaulus Bison MN656991.1 MN503301.1 MT157226.1/ opening in females. Photomicrographs of the features were taken using viviparus bonasus MN656992.1 an Opta-Tech Lab40 light microscope ( × 40 - × 1000 magnification) Dictyocaulus Cervus NC_019809.1 NC_019809.1 NC_019809.1 and the OptaView IS-PL Opta-Tech software package (Opta-Tech, War­ eckerti elaphus saw, Poland). Specimens intended for scanning electron microscopy Angiostrongylus Rattus AP017672.1 AP017672.1 AP017672.1 cantonensis norvegicus (SEM) were dehydrated in increasing concentrations of ethanol and

214 A.M. Pyziel et al. International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220

2.4. Phylogenetic analyses specimens was oval, flattened dorsoventrally with an elongated oval oral opening (Fig. 1A). European bison-derived BCWs were thin (Fig. 1B). Phylogenetic analysis of Dictyocaulus spp. taxa was based on aligned A single ring of four symmetrical submedian cephalic papillae and two amino acid sequences of the combined partial cox1 (654 bp), cytB (714 lateral amphids were observed in the head region (Fig. 1C). Cervical bp) and nad5 (351 bp) nucleotide sequences with the use of the GTR + G papillae were absent, and a nerve ring was present (Fig. 1A and B). Eu­ (cox1 and cytB) and GTR + I (nad5) models of evolution (Table 1). The ropean bison-derived lungworms were longer than the cattle-derived GTR model was chosen on the basis of JModelTest 2.1.10 (Guindon and D. viviparus (Table 2), with the difference being statistically significant Gascuel, 2003; Darriba et al., 2012) using AIC criterion. Phylogenetic for both sexes (p < 0.001 for males and p = 0.029 for females). In addi­ tree was constructed using Bayesian inference (BI) analysis with tion, the BCW was significantlylonger in both the male and female Eu­ MrBayes version 3.2. (Huelsenbeck and Ronquist, 2001). ropean bison lungworm (p < 0.001) (Table 3); however, while the BCWs of males isolated from European bison were significantlynarrower than 2.5. Histopathological examination of the lungs those from cattle (p < 0.001), no such statistically significantdifference was observed for the females (p = 0.176) (Table 3). The European bison The lungs were initially checked for the lungworms directly during and cattle-derived lungworms differed with regard to head width; spe­ dissection conducted under fieldconditions. Fifteen lungs with parasitic cifically,the European bison lungworms presented higher minimum and infection were macroscopically examined, packed into plastic bags and maximum values (Table 3). The female reproductive system of the Eu­ transported immediately to the laboratory. ropean bison lungworm was didelphic-amphidelphic (Fig. 2A). As the Samples of pulmonary tissue surrounding parasites were fixed in anterior/posterior sphincter and infundibulum were only slightly sepa­ 10% buffered formalin and submitted for histopathological investiga­ rated from each other (Fig. 2B), their measurements are given together tion as described in Pyziel et al. (2018a). The samples were dehydrated (Table 4). The female body ended in a tail with two phasmids at its mid- through graded series of ethanol and cleared in xylene, then embedded length (Fig. 2C). The male body ended in a bell-shaped copulatory bursa in paraffinwax, cut into sections (4 μm), stained with hematoxylin and (Fig. 3A and B). A pair of dark brown spicules and gubernaculum was eosin (HE), and processed for light microscope examination (Olympus observed in each male (Fig. 3C). A small transparent membrane around BX43; Olympus, Japan). the tip of each spicule was clearly visible (Fig. 3C). The spicules of Eu­ ropean bison-derived lungworms were significantly longer (p < 0.001) 2.6. Statistical analysis than those isolated from cattle (Table 5).

The measurements for total body length, BCW length, BCW width, 3.2. DNA sequences and spicule length of European bison-derived lungworms taken during this study were compared with those of D. viviparus of cattle presented in Partial nucleotide sequences of mt cox1, nad5, and cytB genes of Divina et al. (2000) with regard to their mean, standard deviation and European bison-derived lungworms were obtained from 15 animals in number of samples. The females and males were separated into groups the Białowieza˙ Primeval and Borecka Forests. Beside one nucleotide and Welch t-test was used to compare those from the European bison difference in a partial sequence of mt nad5, no diversity was found be­ with those from the cattle. It was assumed that the data from the tween the lungworm sequences obtained from these geographical re­ cattle-derived lungworms obtained by Divina et al. (2000) met the as­ gions: the remaining sequences of the mt genes were identical. sumptions for the t-test, as they had been analyzed using a general linear Therefore, only single nucleotide sequences of the cox1 and cytB genes of model. The assumptions regarding the data for Dictyocaulus lungworm European bison worms was submitted from both locations; in contrast, isolated from European bison were also checked. Probability values less three diverse sequences of nad5 were submitted to GenBank. than 0.05 (p < 0.05) were considered statistically significant. Nucleotide diversity was calculated from pairwise sequence align­ ments of partial cox1, cytB and nad5 of D. viviparus from cattle (GenBank: 3. Results JX519460.1; AP017683.1) and from European bison (GenBank: KM359417.1 + KM503300.1 + KM359431.1; MN656991.1 + 3.1. Morphological study MN503301.1 + MT157226.1; MN656991.1 + MN503301.1 + MN656992.1, respectively). These results varied from 1.7% for nad5 (6 Buccal capsule wall (BCW) of investigated European bison-derived nucleotides of difference), and 2.1% for cytB (15 nucleotides of

Fig. 1. Dictyocaulus viviparus of European bison, anterior end. (A) Male, anterior end in optical section, showing head, cephalic vesicle (cv), esophagus, nerve ring (nr), lateral view. (B) Female, anterior end in optical section, showing buccal capsule (bc), buccal capsule wall (bcw), cephalic vesicle (cv), nerve ring (nr), and excretory pore (ep), lateral view. (C) Cephalic region, scanning electron microscopy, showing two lateral amphids (LA) and four submedian papillae (SCP).

215 A.M. Pyziel et al. International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220

Table 2 The length characterization of morphological features of European bison-derived Dictyocaulus viviparus in comparison with cattle-derived lungworm data following Divina et al. (2000). FEATURE HOST

Cattle (Bos taurus) European bison (Bison bonasus) Cattle vs. European bison

male famale male female male female

Bodya Range 14 - 55 14 - 76 20.1–69.5 16.8–83.7 p < 0.001 Sample size (n) n = 37 n = 89 n = 30 n = 35 p = 0.029 Mean ± standard deviation (X ± SD) 38 ± 2 48 ± 1 47.5 ± 11.9 54.4 ± 16.6 Cephalic vesicle Range – – 144.9–378 106.7–283 – Sample size (n) n = 28 n = 9 – Mean ± standard deviation (X ± SD) 242.9 ± 60.2 196.9 ± 67.3 Buccal capsule Range – – 34.1–62 37.8–66 – Sample size (n) n = 11 n = 17 – Mean ± standard deviation (X ± SD) 50 ± 7.9 50.6 ± 7.4 Buccal capsule wall Range 9.9–20.9 6.8–30.9 37 - 53 48.3–84 p < 0.001 Sample size (n) n = 35 n = 77 n = 11 n = 20 p < 0.001 Mean ± standard deviation (X ± SD) 16.2 ± 0.6 16.1 ± 0.4 44.7 ± 4.9 67.1 ± 10.2 Esophagus Range 808–992 931.4–1546 975.5–1562 – Sample size (n) 896 - 1040 n = 26 n = 25 – Mean ± standard deviation (X ± SD) 1281 ± 162.6 1308 ± 172.1 Anterior to nerve ring Range 308–398 235–379 359–592.1 224–612.1 – Sample size (n) – – n = 24 n = 26 – Mean ± standard deviation (X ± SD) – – 479.4 ± 63.2 474.6 ± 84.3 Anterior to excretory pore Range 412–526 393–493 454 - 771 414 - 828 Sample size (n) – – n = 18 n = 19 – – Mean ± standard deviation (X ± SD) – – 656.9 ± 86.2 597.2 ± 107.6

a All dimensions are given in μm except body length, given in mm.

Table 3 The width characterization of morphological features of European bison-derived Dictyocaulus viviparus in comparison with cattle-derived lungworm data following Divina et al. (2000). FEATUREa HOST

Cattle (Bos taurus) European bison (Bison bonasus) Cattle vs. European bison

male famale male female male female

Head Range 74–91 86–104 97.9–166 93.6–191.4 – – Sample size (n) n = 21 n = 23 Mean ± standard deviation (X ± SD) 143.9 ± 17.1 144.6 ± 26.1 Cephalic vesicle Range – – 10 - 42 8 - 31 – – Sample size (n) n = 30 n = 9 Mean ± standard deviation (X ± SD) 23.9 ± 8.2 17.5 ± 8.3 Buccal capsule Range – – 28 - 39 22–48.3 – – Sample size (n) n = 11 n = 16 Mean ± standard deviation (X ± SD) 35.3 ± 3 31.6 ± 7.2 Buccal capsule wall Range 3.9–15.8 2–16.6 4 - 8 4.8–14 p < 0.001 p = 0.176 Sample size (n) n = 35 n = 77 n = 11 n = 24 Non significant Mean ± standard deviation (X ± SD) 8.1 ± 0.4 8.8 ± 0.2 5.8 ± 1.3 8.2 ± 2.1 Esophagus max. Range – – 132.4–365 136.7–287 – – Sample size (n) n = 25 n = 24 Mean ± standard deviation (X ± SD) 247.4 ± 63.1 216 ± 41

a All dimensions are given in μm. difference) to 3.7% for the cox1 marker gene (24 nucleotides of differ­ European bison from Białowieza˙ Primeval Forest (GenBank: ence) (Table 6). Thus, cox1 demonstrated the greatest sequence varia­ KM359417.1 + KM359431.1 + MN503300.1) with their sister taxa from tion, whereas cytB and nad5 were more conserved. At the same time, Borecka Forest - Borki (GenBank: MN656991.1 + MN656992.1/ nad5 was the most diverse gene within European bison-derived worms MT157226.1 + MN503301.1). Another clade included D. viviparus of (0.6%), with up to two nucleotides of difference (Table 6). Additionally, cattle (GenBank: JX519460.1; AP017683.1). A third clade included D. in the analysis of mt sequence variation between D. viviparus (GenBank: sp. cf.eckerti of red (GenBank: NC_019809.1). JX519460.1; AP017683) and D. sp. cf. eckerti (NC_019809.1) the highest level of diversity (18.2%–18.5%) was found for the nad5 region, with 3.4. Macroscopic examination of the lungs the cox1 region being more conserved (11.3%–11.6%) (Table 6). In all cases the lungs were pale pink, airy, pasty and soft. In two cases the lungs were also focally congested. Lungworms were found in bronchi 3.3. Phylogenetic reconstruction and bronchioles of each examined lung, mostly in caudal and cranial lung lobes. They were covered with mucus. The range of infection was Bayesian analysis (BI) of mt sequence data (cox1 + nad5 + cytB) with 12–160 adult lungworms per host. Angiostrongylus cantonensis (GenBank: AP017672.1) as an outgroup revealed two strongly supported clades (Fig. 4). One cluster of Dictyo­ caulus lungworms comprised two subclades: one including taxa of

216 A.M. Pyziel et al. International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220

Fig. 2. Dictyocaulus viviparus of European bison, female genital system, light microscopy. (A) Ovejectors in left lateral view, showing relationships for the vulva (vu), vestibules, and combined anterior infundibulum, and sphincter (ainf + asph), and posterior infundibulum and sphincter (pinf + psph). (B) Region of posterior infundibulum (pinf) and posterior sphincter (psph), left lateral view. (C) female tail, right lateral view, showing anus and phasmids (ph).

Table 4 Dimensional characterization of morphological features of female reproductive system of Dictyocaulus viviparus derived from European bison (own data) in comparison with cattle-derived lungworm data following Divina et al. (2000).

FEATURE HOST

Cattle European bison

Posterior to vulva openinga Range 13–29.5 14.7–41 Sample size (n) – n = 21 Mean ± standard deviation (X ± SD) – 27.3 ± 8.2 Body width at vulva opening Range 322–550 456.9–839.6 Sample size (n) – n = 26 Mean ± standard deviation (X ± SD) – 634.8 ± 93.3 Length of vestibules Range 1255–1996 1165.1–2397 Sample size (n) – n = 15 Mean ± standard deviation (X ± SD) – 1928.4 ± 366.4 Length of anterior sphincter and infundibulum Range 75–114 106 - 175 Sample size (n) – n = 26 Mean ± standard deviation (X ± SD) – 141.2 ± 22.4 Length of posterior sphincter and infundibulum Range 84–113 55 - 171 Sample size (n) – n = 21 Mean ± standard deviation (X ± SD) – 116.5 ± 30.8 Immature eggs in uterus Range 46–60 × 74 - 91 67 - 98,6 × 38.9–60 Sample size (n) – n = 22 Mean ± standard deviation (X ± SD) – 86.8 ± 7.8 × 52.1 ± 5.4 Mature embryonated eggs Range 43–55 × 79 - 86 72.2–99 × 37–48.4 Sample size (n) – n = 9 Mean ± standard deviation (X ± SD) – 84.9 ± 8 × 44.8 ± 3.8 Posterior to anus (length of tail) Range 261–483 323.2–688 Sample size (n) – n = 31 Mean ± standard deviation (X ± SD) – 484.9 ± 65.8 Posterior to phasmids Range – 178 - 286 Sample size (n) n = 29 Mean ± standard deviation (X ± SD) 234.7 ± 12.7

a All dimensions are given in μm except posterior to vulva opening, given in mm.

3.5. Histopathology of the lung tissue the same time hyperplasia of smooth muscle and bronchial epithelial cells was observed, accompanied by the thickening of alveolar septa and The histopathological sections of European bison lung tissue the blood vessel walls (Fig. 5C). Other pulmonary lesions included revealed various lung pathologies associated with Dictyocaulus infection, emphysema and congestion. including numerous cross-sections of mature adult or young adult worms in the lumen of the bronchi and bronchioles (Fig. 5A). Moreover, 4. Conclusion sections of nematode larvae were detected multifocally in pulmonary alveoli (Fig. 5B). Alveolar walls were infiltratedby lymphocytes, plasma Investigated European bison were infected exclusively with cells, macrophages and eosinophils, whereas the bronchi, bronchioles morphologically-different genotype of D. viviparus than cattle. No mixed and blood vessels were surrounded by mononuclear cells (Fig. 5A, C). infections of bison and cattle-derived lungworms was noted in investi­ Histopathological examination revealed lesions consisted with intersti­ gated European bison. We propose, therefore, that the lungworm of tial pneumonia with hyperplasia of the lymphoid follicle (Fig. 5D). At European bison should be classified as D. viviparus subsp. bisontis.

217 A.M. Pyziel et al. International Journal for Parasitology: Parasites and Wildlife 13 (2020) 213–220

Fig. 3. Dictyocaulus viviparus of European bison, male genital system, light microscopy. (A) Bursa, dorsal view. (B) Bursa, left lateral view, showing gubernaculum (gub), and left spicula (spi). (C) Spiculae, dorsal view.

Table 5 Dimensional characterization of morphological features of male reproductive system of Dictyocaulus viviparus derived from European bison in comparison with cattle- derived lungworm data following Divina et al. (2000).

FEATUREa HOST

Cattle European bison Cattle vs. European bison

Length of copulatory bursa Range – 205.7–415 – Sample size (n) 25 Mean ± standard deviation (X ± SD) 310.5 ± 59.1 Width of gubernaculum Range – 26.7–44 – Sample size (n) 19 Mean ± standard deviation (X ± SD) 33.4 ± 4.6 Length of spicules Range 100.1–286.9 271.5–385 p < 0.001 Sample size (n) 64 57 Mean ± standard deviation (X ± SD) 209.7 ± 15.4 310.8 ± 18.8

a All dimensions are given in μm.

′′ 4.1. Taxonomic summary 1214 E) Specimens deposited: Holotype (male) and allotype (female): MNHW The specific epithet: D. viviparus subsp. bisontis. 1353 (Museum of Natural History, Wroclaw University, Poland) Type and only known host: European bison, Bison bonasus (Linnaeus, Sequences deposited: GenBank: KC771250.1, KF007338.1 – 1758) (Artiodactyla: Bovidae) KF007341.1, KM359411.1 - KM359435.1, KT581635.1 - KT581637.1, Site of infection: Trachea, bronchi, bronchioli MT157226.1, MN503300.1, MN503301.1, MN656991.1, MN656992.1. Type locality: Białowieza˙ Primeval Forest, podlaskie voivodship, Etymology: The specific epithet is derived from host genus name. ◦ ′ ◦ ′ ◦ ′ ◦ ′ Poland (52 29 N - 52 57 N, 23 31 E 24 21 E), Borecka Forest, war­ ◦ ′ ′′ ◦ ′ minsko-mazurskie´ voivodship, Poland (54 08 01, 9850 N, 22 05 39,

Table 6 Pairwise comparison of sequence alignment (1719 bp) consisted of cox1 (654 bp), cytB (714 bp) and nad5 (351 bp) mt DNA nucleotide sequence and inferred amino acid sequence (alignment: 573; cox1: 218; cytB: 238; nad5: 117) variability among Dictyocaulus eckerti and isolates of D. viviparus of cattle and European bison. Above diagonal = Number of variable sites in the 1719 base pair (bp). Below diagonal = Number of variable sites in the 573 amino acid. Total number of variable sites between 2 species/isolates is given prior to parenthesis, whereas number of variable sites regarding particular marker genes (cox1, cytB and nad5, respectively) are given in parenthesis.

Species and hosta 1 2 3 4 5 6

1. D. eckerti, red deer 258 (76; 115; 68) 259 (76; 115; 67) 258 (76; 115; 67) 256 (74; 117; 65) 258 (76; 118; 64) 2. D. viviparus, European bison 51 (5; 29; 17) 1 (0; 0; 1) 0 45 (22; 15; 8) 46 (24; 15; 7) 3. D. viviparus, European bison 51 (5; 29; 17) 0 1 (0; 0; 1) 44 (22; 15; 7) 45 (24; 15; 6) 4. D. viviparus, European bison 51 (5; 29; 17) 0 0 45 (22; 15; 8) 46 (24; 15; 7) 5. D. viviparus, cattle 52 (6; 28; 18) 6 (1; 4; 1) 6 (1; 4; 1) 6 (1; 4; 1) 5 (2; 2; 1) 6. D. viviparus, cattle 52 (6; 28; 18) 6 (1; 4; 1) 6 (1; 4; 1) 6 (1; 4; 1) 0

a GenBank: 1 = NC_019809.1; 2 = KM359417.1 + KM503300.1 + KM359431.1; 3 = MN656991.1 + MN503301.1 + MT157226.1; 4 = MN656991.1 + MN503301.1 + MN656992.1; 5 = JX519460.1; 6 = AP017683.1.

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Fig. 4. Phylogenetic tree of Dictyocaulus spp. based on cox1, cytB and nad5 partial sequences, constructed with the use of Bayesian inference (BI) analysis using MrBayes version 3.2. The GTR + G (cox1, cytB) and GTR + I (nad5) model was chosen based on jMo­ delTest version 2.1.4 using Akaike Information Cri­ terion. The analysis was run for 2,000,000 generations, with 500,000 generations discarded as ‘burn-in’. Hosts (for Dictyocaulus viviparus) and Gen­ Bank accession numbers of origin are shown. Nodal support is indicated as Bayesian posterior probabili­ ties. Sequence from Angiostrongylus cantoniensis (AP017672.1) was used as an outgroup.

Fig. 5. Histopathological lesions in the lungs of European bison infected with Dictyocaulus viviparus. (A) The cross-section of a nematode in the lumen of bronchiole (arrow); mononuclear cells infiltrationin the mucous membrane of the bronchiole. H-E stain ( × 40 magnification).(B) Multiple cross-sections of scattered within alveoli and bronchiole (arrows). H-E stain ( × 40 magnification). (C) Interstitial pneumonia, diffuse, lymphoplasmacytic, eosinophilic with the hyperplasia of the vascular wall (arrows). H-E stain ( × 40 magnification).Inset: Perivascular, peribronchiolar lymphocytic and eosinophilic infiltration; on the left side: the tunica media of vessels is thickened (hyperplastic). H-E stain ( × 200 magnification). (D) Prominent peri­ bronchiolar hyperplasia of the lymphoid follicle (arrow); pulmonary tissue showed interstitial pneumonia pattern. H-E stain ( × 200 magnification).

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5. Discussion between the genotypes of the lungworms infecting cattle and bison.

This study provides a detailed morphological description of Euro­ Acknowledgment pean bison-derived Dictyocaulus lungworms, and assess the phylogenetic position of lungworms among closely-related hosts: European bison and Presented data were generated during a scientific stay at the SLU domestic cattle. According to Divina et al. (2000), the most important supported by the Warsaw University of Life Sciences (WULS-SGGW) morphological diagnostic characteristics for Dictyocaulus spp. are BCW within the framework of the WULS-SGGW Own Scholarship Found thickness and length, while spicular length does not differ between (Własny Fundusz Stypendialny Szkoły Głownej´ Gospodarstwa Wiej­ cattle, roe deer and moose-derived lungworms. In the present study, the skiego w Warszawie), decision nr. BWM – 315/2018. The cost of the specimens from the European bison were found to have longer BCW than study was coved by Petra Lundbergs Stiftelse (2018). those isolated from cattle: this different was almost three-fold in males The authors would like to express their gratitude to mgr Ed Low­ and more than four-fold in females; in addition, the BCW was narrower czowski for English correction of the text. in the males of European bison than in those diagnosed in cattle. In addition, like the majority of cattle-derived lungworms, the BCW of References European bison lungworm was classified as thin (Divina et al., 2000). Furthermore, the spicules of Dictyocaulus were significantly longer in Blouin, M.S., 1998. Mitochondrial DNA diversity in nematodes. J. Helminthol. 72, 285–289. European bison than those of cattle (Divina et al., 2000). Divina et al. Blouin, M.S., 2002. Molecular prospecting for cryptic species of nematodes: (2000) also report that the thickness and length of BCW were positively mitochondrial DNA versus internal transcribed spacer. Int. J. Parasitol. 32, 527–531. correlated with the total body length of the lungworm. Our morpho­ Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. JModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. logical findingssuggest that European bison host a distinct Dictyocaulus Demiaszkiewicz, A.W., Pyziel, A.M., Lachowicz, J., Kuligowska, I., 2009. Dictyocaulosis species; however, it is well known that morphological differences of European bison in Białowieza˙ forest. European Bison Conserv. News. 2, 112–118. (morphotypes) can be induced by the host species (Nadler, 2002). Divina, B.P., Wilhelmsson, E., Mattsson, J.G., Waller, P., Hoglund,¨ J., 2000. Nevertheless, although our present results suggest that Dictyocaulus Identification of Dictyocaulus spp. in ruminants by morphological and molecular analyses. Parasitology 121, 193–201. lungworms of European bison and cattle can be morphologically Eisenback, J.D., 1985. Techniques for preparing nematodes for scanning electron distinguished on the basis of the BCW length, the total body length, and microscopy. In: Barker, K.R., Carter, C.C., Sasser, J.N. (Eds.), An Advanced Treatise spicule lengths in males, their phylogenetic position suggests that cattle on Meloidogyne. North Carolina State University, Raleigh, North Carolina, pp. 79–105. lungworm can infect both hosts. This suggestion is supported by previ­ Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for ous findingsinvolving rDNA datasets, showing that the SSU sequence of amplification of mitochondrial cytochrome c oxidase subunit I from diverse – European bison lungworms (GenBank: KC771250) was homologous metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294 299. Gasser, R.B., 2006. Molecular tools – advances, opportunities and prospects. Vet. with those from cattle (GenBank: AY168856) (Pyziel, 2014); addition­ Parasitol. 136, 69–89. ally, both isolates diverged only slightly in the ITS2 region (GenBank: Gasser, R.B., Jabbar, A., Mohandas, N., Hoglund,¨ J., Hall, R.S., Littlewood, D.T.J., Jex, A. KM359413; KM359411; U37718; AF105257) (Pyziel et al., 2017). Pre­ R., 2012. Assessment of the genetic relationship between Dictyocaulus species from Bos taurus and Cervus elaphus using complete mitochondrial genetic datasets. vious studies have found mt DNA sequences to be more variable within a Parasites & Vectors Parasite Vector 5, 241. nematode species than rDNA (Gasser, 2006; Gasser et al., 2012), and Guindon, S., Gascuel, O.A., 2003. Simple, fast, and accurate algorithm to estimate large hence mt marker genes are more suitable for studying population ge­ phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hu, M., Hoglund,¨ J., Chilton, N.B., Zhu, X., Gasser, R.B., 2002. Mutation scanning netics and systematics in this regard (Blouin, 2002; Gasser et al., 2012). analysis of mitochondrial cytochrome c oxidase subunit 1 reveals limited gene flow Current and previous comparisons of the mt DNA from lungworms from among bovine lungworm subpopulations in Sweden. Electrophoresis 23, 3357–3363. cattle and red deer (Gasser et al., 2012; Pyziel et al., 2017, 2018b) found Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogenetic trees. – cox1 to capture the least within-genus sequence variation and nad5 the Bioinformatics 17, 754 755. Hoglund,¨ J., Morrison, D.A., Mattsson, J.G., Engstrom,¨ A., 2006. Population genetics of greatest. Similarly, cox1 was found to demonstrate the least the bovine/cattle lungworm (Dictyocaulus viviparus) based on mtDNA and AFLP within-species variation of mtDNA genes among populations of marker techniques. Parasitology 133, 89–99. D. viviparus isolated from cattle (Hoglund¨ et al., 2006; Gasser et al., IUCN, 2020. Red list of threatened species. https://www.iucnredlist.org/. (Accessed 21 August 2020). 2012). These findings contrast with those obtained from cattle and Eu­ Keller, L.F., Waller, D.M., 2002. Inbreeding effects in wild populations. Trends Ecol. Evol. ropean bison-derived lungworms in the present study, which found nad5 17, 230–241. and cytB to be more conserved than cox1. In addition, the worms from Klich, D., Łopucki, R., Stachniuk, A., Sporek, M., Fornal, E., Wojciechowski, M., Olech, W., 2020. Pesticides and conservation of large ungulates and conservation of both hosts demonstrated comparable levels of sequence diversity for the large ungulates: health risk to European bison from plant protection products as a same gene regions to those noted previously between various result of crop depredation. Polos One 15, e0228243. D. viviparus isolated from cattle; this diversity did not reach 4.0% (Hu Mahmood, F., Khan, A., Hussain, R., Anjum, M.S., 2014. Prevalence and pathology of ¨ Dictyocaulus viviparus infection in cattle and buffaloes. J. Anim. Plant Sci. 24, et al., 2002; Hoglund et al., 2006; Gasser et al., 2012). In contrast, mt 743–748. sequence variation has been found to range from 10 to 20% between Nadler, S., 2002. Species delimitation and nematode biodiversity: phylogenies rule. closely-related species of nematodes and from 1 to 3% between in­ Nematology 4, 615–625. Pyziel, A.M., 2014. Molecular analysis of lungworms from European bison (Bison bonasus) dividuals of inbreeding population (Blouin, 1998). on the basis of small subunit ribosomal RNA gene (SSU). Acta Parasitol. 59, 122–125. Our findings also indicate that lungworm infection can cause inter­ Pyziel, A.M., Laskowski, Z., Demiaszkiewicz, A.W., Hoglund,¨ J., 2017. Interrelationships stitial pneumonia, bronchitis and bronchiolitis. Similar histopatholog­ of Dictyocaulus spp. in wild ruminants with morphological description of Dictyocaulus ical lesions have been observed in the lung tissue of cattle and buffaloes cervi sp. (Nematoda: ) from red deer, Cervus elaphus. J. Parasitol. 103, 506–518. (Schnider et al., 1991; Mahmood et al., 2014) exhibiting parasitic Pyziel, A.M., 2018. Applicability of selected ribosomal and mitochondrial genetic pneumonia with extensive mononuclear infiltration in the lung paren­ markers in identification of European bison lungworm: a state of the art review. – chyma, multifocal lymphocytic and eosinophilic bronchiolitis. Addi­ European Bison Conserv. News. 11, 31 38. Pyziel, A.M., Dolka, I., Werszko, J., Laskowski, Z., Steiner-Bogdaszewska, Z.,˙ tionally, the histopathological picture of European bison dictyocaulosis Wi´sniewski, J., Demiaszkiewicz, A.W., 2018a. Pathological lesions in the lungs of red revealed the presence of similar lesions to those noted previously in red deer Cervus elaphus (L.) induced by a newly-described Dictyocaulus cervi (Nematoda: deer dictyocaulosis (Pyziel et al., 2018a). trichostrongyloidea). Vet. Parasitol. 261, 22–26. Pyziel, A.M., Laskowski, Z., Hoglund,¨ J., 2018b. An assessment of the use of cox1 and To summarise, it appears that D. viviparus infecting cattle and European cox3 mitochondrial genetic markers for the identification of Dictyocaulus spp. bison are characterized by both morphological differences and slightly (Nematoda: trichostrongyloidea) in wild ruminants. Parasitol. Res. 117, 2341–2345. diverging genotypes. We therefore propose that the lungworm of European Radwan, J., Biedrzycka, A., Babik, W., 2010. Does reduced MHC diversity decrease viability of vertebrate populations? Biol. Conserv. 143, 537–544. bison should be classified as D. viviparus subsp. bisontis. An additional Schnider, T., Kauo, F.J., Drommer, W., 1991. Morphological investigations on the recommendation is that mt cytB marker gene should be used to distinguish pathology of Dictyocaulus viviparus infections in cattle. Parasitol. Res. 77, 260–265.

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